Cloning and Characterization of thePseudomonas fluorescens ATP-Binding Cassette Exporter, HasDEF, for the Heme Acquisition Protein HasA

摘要

Two ATP-binding cassette (ABC) exporters are present inPseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa andSerratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens andP. aeruginosa but not S. marcescens HasA inEscherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. TheP. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens andS. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.